Cartridges and Instruments for Sample Analysis

ABSTRACT

Provided herein are instruments and cartridges for processing samples. The cartridges include fluidic circuits in which fluid movement can be regulated by diaphragm valves. In certain cartridges, deformable material providing a diaphragm contacts an interface in the instrument that actuates the diaphragm directly, without intervening actuation layer. Certain cartridges have a plurality of fluidic circuits and fluid distribution channels or pneumatic distribution channels configured to deliver fluids or pneumatic pressure to any of the fluidic circuits, selectively. Certain cartridges have compartments containing on-board reagents. Compartments can be closed by a film attached to a body the cartridge through a heat seal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/258,412, filed Jan. 25, 2019, which is a divisional of U.S.application Ser. No. 15/037,039, filed May 16, 2016, now U.S. Pat. No.10,191,071, which is a nationalization of PCT Application No.PCT/US14/66008, filed Nov. 17, 2014, which claims the benefit of andpriority to U.S. Provisional Application No. 61/905,804 filed Nov. 18,2013 and U.S. Provisional Application No. 61/981,678, filed Apr. 14,2014, each of which is incorporated herein by reference in its entirely.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

None.

BACKGROUND OF THE INVENTION

One barrier to the broad adoption of rapid DNA-based humanidentification is the consumable cost. A low-cost cartridge using smallamounts of reagents would reduce this barrier. Previous approaches havefocused on automation and manufacturing process improvement to reducethe cost to make a given cartridge.

Versions of systems including sample cartridges and fluidic systems forsample extraction and analysis are described in, for example, U.S. Pat.No. 6,190,616 (Jovanovich et al.); U.S. Pat. No. 6,551,839 (Jovanovichet al.); U.S. Pat. No. 6,870,185 (Jovanovich et al.); U.S. Pat. No.7,244,961 (Jovanovich et al.); U.S. Pat. No. 7,445,926 (Mathies et al.);U.S. Pat. No. 7,799,553 (Mathies et al.); U.S. Pat. No. 8,173,417 (Tanet al.); U.S. Pat. No. 8,206,974 (Tan et al.); U.S. Pat. No. 8,394,642(Jovanovich et al.); U.S. Pat. No. 8,425,861 (Selden et al.); U.S. Pat.No. 8,431,340 (Jovanovich et al.); U.S. Pat. No. 8,720,036 (Selden etal.) and U.S. Pat. No. 8,858,770 (Tan et al.); US patent applications2009/0178934 (Jarvius); 2009/0253181; 2011/0039303 (Jovanovich et al.);2011/0126911 (Kobrin et al.); 2011/0220502 (Selden et al.); 201 2/01 81460 (Eberhart et al.); 2013/0139895 (Vangbo) and 2013/0115607 (Nielsenet al.); and International Patent Application WO/US2013/130910.

SUMMARY OF THE INVENTION

Fluidic devices are provided, for example in the form of a cartridge,for sample extraction and analyte reaction and analysis.

Provided herein is a cartridge comprising one or more fluidic circuitsthat each comprise at least one diaphragm valve; wherein the cartridgecomprises: (a) a body comprising (i) a surface comprising a valve seatin fluidic communication with a valve inlet and a valve outlet and (ii)at least one port in fluidic communication with the fluidic circuit; and(b) a layer of deformable material covering the valve seat and the atleast one port, wherein a portion of the layer of deformable materialfunctions as a diaphragm which, in combination with the valve seat,forms a diaphragm valve, and wherein the diaphragm, when actuated (forexample, by being moved into contact with the valve seat or by beingmoved out of contact with the valve seat), regulates fluid flow acrossthe diaphragm valve, and comprising at least one conduit through thelayer of deformable material, each conduit communicating with a port;and wherein the cartridge is configured to engage a cartridge interface,putting the deformable material in direct contact with a surface ofcartridge interface, putting the diaphragm in communication with asource of positive and/or negative pressure that actuates the diaphragm(e.g., pneumatically or mechanically) and putting the at least one portin communication with a fluid or pneumatic line through the conduit,wherein the layer of deformable material optionally functions as agasket sealing the at least one port against leakage. In one embodimentthe fluidic circuit further comprises a reaction chamber formed in thebody, optionally covered with a film of heat conductive material (e.g.,a metal film). In another embodiment the fluidic circuit furthercomprises a chamber configured to receive a sample comprising abiological material, said chamber optionally comprising a close tab. Anexample of such an instrument and cartridge is shown in FIGS. 1-5.

Also provided herein is an instrument comprising: (a) at least onecartridge interface comprising: (i) an engagement unit configured toreceive a removably insertable cartridge and to engage a receivedcartridge with a manifold assembly and, optionally, a thermal regulator,wherein the cartridge comprises at least one fluidic circuit comprisingat least one fluid channel, at least one exit port; and at least onediaphragm valve comprising a valve seat configured to regulate fluidflow in the at least one fluidic circuit; and a layer of deformablematerial covering the valve seat and the at least one port, wherein thedeformable material functions as a diaphragm in the diaphragm valve andcomprises at least one conduit through the layer, each conduitcommunicating with a port; and (ii) a manifold assembly comprising anengagement surface configured to directly contact the deformable layerof a received cartridge and having a plurality of transmission channelscommunicating with ports on the engagement surface; wherein engaging acartridge with the manifold assembly: (A) puts the diaphragm incommunication with a first port on the engagement surface configured totransmit positive or negative pressure to the diaphragm, and (B) putsthe exit ports in communication with second ports on the engagementsurface configured to transmit fluid into or out of a fluidic circuitthrough the transmission channels; and (iii) a thermal regulator which,when engaged with a received cartridge, puts a heat pump (e.g., athermoelectric heater, e.g., a Peltier device) in thermal contact with athermal cycling chamber in the cartridge or a heating element in thermalcontact with a chamber in the cartridge. In one embodiment theinstrument of further comprises any of: (b) a pneumatic pressure sourceconfigured to deliver positive or negative pneumatic pressure to atransmission channel of the manifold assembly; (c) a pump configured tomove liquid into or out of a transmission channel of the manifoldassembly; (d) a source of reagents in fluid communication with atransmission channel of the manifold assembly; (e) an analysis moduleconfigured to perform at least one analysis (e.g., electrophoresis) on afluid received from the cartridge; and (f) a control module comprisingexecutable code that, when executed, controls operation of theinstrument.

Also provided herein is a cartridge comprising a first layer and adeformable layer: (a) wherein the first layer comprises: (i) a firstside contacting the deformable layer, wherein the first side comprises aplurality of fluidic circuits, each fluidic circuit comprising at leastone fluidic channel; and (ii) a second side comprising at least onefluidic distribution channel, which fluidic distribution channel iscovered by a cover layer; (iii) a plurality of vias in the first layer,each via configured to put the fluidic distribution channel incommunication with a fluidic channel; and (iv) optionally, at least onevia in the first layer configured to put the fluidic distributionchannel in communication with a port on a side of the first layerconfigured to engage a source of fluid; and (b) optionally comprising anactuation layer comprising at least one actuation circuit configured toactuate a diaphragm against a valve seat in the first side. An exampleof such a cartridge is shown in FIGS. 6 and 9.

Also provided herein is a cartridge comprising a first layer, a secondlayer and a deformable layer sandwiched there between: (a) wherein thefirst layer comprises: (i) a first side contacting the deformable layer,wherein the first side comprises a plurality of fluidic circuits, eachfluidic circuit comprising at least one fluidic channel and at least onevalve seat; (ii) a second side comprising at least one pneumaticdistribution channel, which pneumatic distribution channel is optionallycovered by a cover layer; (iii) at least one via in the first layerconfigured to put a pneumatic distribution channel in communication withthe deformable layer; and (iv) optionally, at least one via in the firstlayer configured to put a pneumatic distribution channel incommunication with a port on a side of the first layer configured toengage in a source of pneumatic pressure; (b) wherein portions of thedeformable layer, in combination with valve seats in the first layerform diaphragm valves; and (c) wherein the actuation layer comprises atleast one actuation circuit comprising at least one branch channel,wherein each branch channel is configured to actuate a diaphragm againsta valve seat in a different fluidic circuit and further comprises avalve seat; and wherein positive or negative pressure applied to apneumatic distribution channel transmits pressure through the via toactuate a diaphragm portion of the deformable layer into or out ofcontact with the valve seat in the branched channel, wherein closing thecontrol valve inhibits actuation the diaphragm valve in the fluidiccircuit. An example of such a configuration is shown in FIG. 10A-B.

Also provided herein is a cartridge comprising: (a) a body comprising apolymer and comprising at least one functional feature on a surface ofthe body and configured to transmit fluid (e.g., a port, a via, a fluidchannel, a chamber, a valve inlet and valve outlet and/or a valve seat);and (b) a layer of deformable material thermally bonded to the body andcovering the functional feature (optionally, wherein at least oneportion of the layer comprises a permanent deformation), and whereinapplication of positive or negative pressure to at least one portion ofthe layer actuates the deformable material into or out of contact with afunctional feature on the surface of the body; and wherein the cartridgeis configured to engage a cartridge interface configured to supplypositive or negative pressure to the at least one portion of the layer.In one embodiment the cartridge further comprises a fluid-filled chamberin the body, wherein the chamber has an opening sealed closed with aheat seal material. In another embodiment the deformable materialcomprises a heat seal material. In another embodiment the deformablematerial comprises a material selected from polypropylene, polyethylene,polystyrene, cycloolefin co-polymer (COC), mylar, polyacetate and ametal. An example of valves in such a cartridge is shown in FIGS. 7, 8,13, and 14 (seal not shown in all figures).

Also provided herein is a device comprising the aforementioned cartridgeand a ram configured to actuate a diaphragm of a diaphragm valve on thebody.

Also provided herein is a device comprising a cartridge and a clampingdevice: (a) wherein the cartridge comprises: (i) a body comprising: (A)at least one fluidic circuit comprising: (I) at least one functionalfeature on a surface of the body and configured to transmit fluid (e.g.,a port, a fluid channel, a chamber, a valve inlet and valve outletand/or a valve seat), wherein the functional feature optionallycomprises a ridge on the surface of the body; and (II) at least onecompartment containing a liquid, wherein the compartment communicateswith the functional feature through one or more vias in the body; and(ii) a layer of deformable material covering the functional feature; and(b) wherein the clamping device, when engaged with the cartridge,applies sufficient pressure to the deformable material to deform thedeformable material against the cartridge body and seal against movementof liquid from the compartment and through the at least one functionalfeature; and wherein removing the clamping device releases pressure tothe deformable material, allowing the seal to open (e.g., through anelastic response of the deformable material or through application ofpositive or negative pressure against the seal). In one embodiment theclamping device comprises a mechanical clamp or a vacuum seal. Anexample of such a cartridge is shown in FIGS. 6 and 11.

Also provided herein is a cartridge comprising: (a) a fluidics layercomprising a surface having at least one diaphragm valve comprising avalve seat (e.g., a recessed valve seat); (b) a deformable layer matedto the surface, wherein a portion of the deformable layer functions as adiaphragm which, when actuated, is configured to move into or out ofcontact with the valve seat; and wherein the portion of the deformablelayer functioning as a diaphragm comprises a boss positioned on a sideof the deformable layer opposite of a side that contacts the valve seat;and (c) optionally comprising: a rigid substrate mated with thedeformable layer and comprising apertures exposing the bosses andconfigured to receive a ram that contacts the boss and actuates thediaphragm, e.g., by application of mechanical pressure; or configured toengage an interface comprising apertures exposing the bosses andconfigured to receive a ram that contacts the boss and actuates thediaphragm, e.g., by application of mechanical pressure. An example ofsuch a cartridge is shown in FIGS. 15 A and B.

Also provided herein is a cartridge comprising: (a) a base comprising:(I) a central barrel comprising a pump chamber and movable syringe, (II)a base floor comprising a port station comprising a floor port; and(Ill) a channel fluidically connecting the barrel chamber to the port inthe floor; and (b) a turret configured to revolve around the centralbarrel and comprising a plurality of turret chambers, each turretchamber comprising a turret chamber aperture in a chamber floor of theturret chamber and facing the base floor, wherein positioning a turretchamber at the port station puts the turret chamber aperture in fluidcommunication with the barrel chamber through the floor port, andwherein the floor closes a turret chamber aperture when the turretchamber is positioned at at least one position other than the portstation; and wherein at least one turret chamber further comprises achannel communicating between the floor port and an exit port. Anexample of such a cartridge is shown in FIG. 17.

Also provided herein is a instrument comprising a cartridge interfaceand a removable cartridge engaged therewith: (a) wherein the interfacecomprises a base and one or more hollow bore pins for delivering fluidto a port in the cartridge, wherein the pin is biased against the baseby the cartridge and protrudes through an aperture in the base; andwherein the pin comprises a home lead-in configured to put the pin in ahome position after the bias is released by removal of the cartridge;and a tapered end configured to mate with a guide in the cartridge; and(b) wherein the cartridge comprises a fluidic circuit comprising a portand a pin guide communicating with the port, wherein the pin guidecomprises a mating cone configured to mate with a tapered end of the pinand a pin guide lead-in configured to guide the pin into the mating conewhen the cartridge exerts a bias against the tapered end of the pinduring engagement with the interface. An example of such a combinationis shown in FIG. 18.

Also provided herein is a cartridge comprising: (a) a body comprising atleast one puncturing element, at least one fluidic channel and at leastone flange; and (b) a reagent reservoir comprising at least onefluidically isolated, fluid filled compartment; a breakable seal in awall of the compartment and a breakable tab attached to an outside wallof the compartment; wherein the cartridge is configured such that whenthe reagent reservoir is engaged with the body: (i) the puncturingelements punctures the breakable seal, putting the compartment influidic communication with the channel; and (ii) the flange exerts aforce against the breakable tab, breaking the tab and forming a vent inthe compartment. An example of such an embodiment is shown in FIG. 12.

Also provided herein is a fluidic device comprising one or morediaphragm valves, each diaphragm valve configured to regulate fluid flowin a fluidic channel, wherein the fluidic device comprises a fluidicslayer, an actuation element and a deformable membrane, wherein eachdiaphragm valve comprises: a) a diaphragm comprised in the deformablemembrane; b) a valve seat comprised in the fluidics layer and recessedfrom a surface of the fluidics layer that contacts the deformablemembrane so that the diaphragm does not close the diaphragm valve unlesspositive pressure is exerted on the diaphragm; and c) a valve inlet anda valve outlet comprised in the fluidics layer and in fluidcommunication with a fluidic channel; and d) a ram configured to actuatethe diaphragm; wherein the deformable membrane comprises a plasticmaterial adhered to the fluidics layer through a heat activatedadhesive, thermal fusion, chemical bonding or a pressure sensitiveadhesive, and wherein the deformable membrane is configured such thatpressure on the deformable membrane presses the membrane against thevalve seat, thereby closing the valve and wherein the valve can beopened by releasing pressure of the ram against the deformable membraneor by pushing liquid through the valve. In one embodiment the deformablemembrane is not an elastomeric material, e.g., is not PDMS. In anotherembodiment the deformable membrane comprises a laminate comprising theplastic material and a deformable, space-filling material, whereinpressure against the space-filling material causes the space fillingmaterial to fill the valve chamber sufficiently so that of the plasticmaterial closes the valve. In another embodiment the deformable materialhas a durometer value of between 10 to 80 Shore D. In another embodimentthe deformable material has a thickness sufficient such that themechanical pressure is applied the deformable material sufficientlyfills the valve chamber to form a seal between the plastic material in avalve seat to close the valve. In another embodiment the deformable,space-filling material comprises a solid foam. In another embodiment thedeformable material is attached to the plastic material through anadhesive. In another embodiment the deformable material is pressed intocontact with the plastic layer through an interface device. In anotherembodiment the fluidics layer comprises a polymer, e.g., athermoplastic.

Also provided herein is a fluidic device comprising one or morediaphragm valves, each diaphragm valve configured to regulate fluid flowone or more fluidic channels, wherein the fluidic device comprises afluidics layer, an actuation element and a deformable membrane, whereineach diaphragm valve comprises: a) a diaphragm comprised in thedeformable membrane; b) a valve seat comprised in the fluidics layer andrecessed from a surface of the fluidics layer that contacts thedeformable membrane so that the diaphragm does not close the diaphragmvalve unless positive pressure is exerted on the diaphragm; and c) avalve inlet and a valve outlet comprised in the fluidics layer and influid communication with a fluidic channel; and d) a ram comprised as anactuation element having a forked end comprising tines, wherein the endhas a surface complying with the valve seat and wherein the tines arecompliant to lateral pressure whereby pressure by the ram on thediaphragm and against the valve seat closes the valve. Embodiments areshown in FIGS. 29-32.

Also provided herein is a fluidic device comprising a sample input, asample output and a waste chamber, all fluidically connected throughfluid channels wherein the waste chamber comprises a material thatdegrades nucleic acid. In one embodiment the material that degradesnucleic acid comprises a hypochlorite salt. In another embodiment thematerial that degrades nucleic acid comprises an enzyme such as anexonuclease or an endonuclease.

Also provided herein is a fluidic device comprising a fluidic circuitcomprising sample input, a reaction chamber and a sample output, whereinthe reaction chamber comprises a solid substrate, e.g., solid phaseextraction material, for retaining analyte from a sample. In oneembodiment the solid substrate comprises a material that binds nucleicacid. In another embodiment the solid substrate comprises Whatman FTApaper, a carboxylated material, a sponge-like material, a polymermembrane, or glass particles. In another embodiment the solid substratebinds a predetermined amount of material. Embodiments are shown in FIGS.22-28.

Also provided herein is a method comprising 1. A method comprising: (a)providing a reaction mixture comprising: (I) a sample comprisingmammalian (e.g., human) DNA, (II) reagents for amplifying short tandemrepeats (STRs) in the mammalian DNA (e.g., labeled primers, nucleotidesand polymerase) and (III) a mammalian-specific probe selected to beamplified in the reaction and including a label that is distinguishablefrom the labeled primers; (b) performing an STR reaction comprisingamplifying STRs in the sample and the mammalian specific probe; (c)detecting an amount of amplified mammalian specific probe in thereaction, e.g., over time, e.g., performing real-time PCR; and (d)optionally, stopping the STR reaction based on the amount of amplifiedmammalian specific probe detected. In one embodiment the labels arefluorescent labels and the distinguishable label has a wavelength aboveor below the highest or lowest wavelength of labeled primers. In anotherembodiment the mammalian specific probe further comprises a quenchersuch as a Black Hole Quencher® or a TaqMan® probe.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrative claims,in which the principles of the disclosure are utilized, and theaccompanying drawings of which:

FIG. 1 shows an instrument 101 comprising an interface adapted to engagea cartridge, including a cartridge interface 103 and a cartridge 200inserted into a slot 107. The embodiment shown contains fourcartridge-receiving subassemblies and Peltier device 109.

FIG. 2 shows cartridge 200, which is insertable into instrument 101. Thecartridge includes a body 201. It further includes elastic layer 203attached to a surface of the body. Layer 203 provides a diaphragm for adiaphragm valve on a surface of body 201, as well as ports 215communicating with fluidic channels in body 201. The cartridge alsoincludes cover sheet 205 that seals a chamber in the body and/orfunctions to transmit heat to or from the chamber. For example, when thecartridge is engaged with the interface, the chamber can function as athermal cycling chamber and cover sheet 205 can be in contact with asource of thermal energy, such as a Peltier device 109. The cartridgealso includes a slot 207 adapted for receiving a sample. Aperture 209and notch 225 are alignment features configured to align cartridge 200with cartridge interface 103. The cartridge also includes a closable cap211.

FIG. 3 shows a cutaway view of cartridge 200. Reaction chamber 301 is inthermal contact with the cover sheet 205 which can be sealed to thebody. Cartridge 200 includes diaphragm valve 303. Diaphragm valve 303includes recessed valve seat 305 defining a valve chamber. Valve inlet306 and valve outlet 307 are configured as vias through body 201 andcommunicate with fluidic channels in the body. Deformable layer 203includes a portion functioning as a diaphragm 311. Putting diaphragm 311into contact with valve seat 305 closes diaphragm valve 303. Deformablelayer 203 also comprises port 309 that communicates with a fluidicconduit in the cartridge. When the cartridge is engaged with a cartridgeinterface, deformable layer 203 functions as a gasket that seals aroundport 309.

FIG. 4 shows from one aspect an exploded view of a cartridge 200 and anassembly 401 of a cartridge interface. The cartridge includes body 201,cover sheet 205, and deformable layer 203. The body includes, on oneside, fluidic channel 213 and reaction chamber 301. Fluidic channelscommunicate through apertures in body 201 with a face covered bydeformable layer 203. The deformable layer 203 includes ports 215configured to align with ports 403 on the interface assembly 401 and anarea 311 positioned to function as a diaphragm in a diaphragm valve.Fluidic lines 405 connect to interface assembly 401 and transmit fluidsto port 403 which connects to cartridge 201. Pneumatic line 407 alsoconnects to interface assembly 401 and transmits positive or negativepneumatic pressure to port 409 which actuates diaphragm for 311.

FIG. 5 shows from another aspect an exploded view of a cartridge of thisdisclosure and an assembly 401 of a cartridge interface. Body 201includes a valve body with valve seat 305 with apertures 306 and 307which is covered by deformable layer 203. Interface assembly (401)includes pneumatic line 407 that, when engaged with deformable layer203, transmits positive or negative pressure to actuate diaphragm 311.Interface assembly 401 also includes fluid lines 405 communicatingthrough passages in interface 401 with apertures 215 in the deformablelayer 203 to allow movement of fluids into, through an out of fluidiccircuits in the cartridge.

FIG. 6 shows cartridge 601 comprising a plurality of sample receptacles607 and comprising reagent chambers in piece 905, distribution channels903 that distribute reagents from reservoirs 603 across a plurality offluidic circuits. Piece 905 includes reagent chambers 1107. Piece 905 iscovered with deformable layer 907. Clamping elements 1103 apply pressureto deformable layer 907. Deformation of the deformable layer againstpiece 907 blocks movement of liquid through passages. This preventsmovement of reagents through fluidic circuits during shipping.

FIG. 7 shows a cartridge 701 comprising a body 1205 having, on side1311, fluidic channel 1315 and valve seat 1317. On another side, thecartridge has a reagent container having open compartments 1201 that cancomprise a seal of a layer of material, for example through a heat seal,to fluidically isolate fluids contained in the compartments until use.

FIG. 8 shows a cartridge 801 comprising a body having a first sidehaving open compartments 811, ports 813 and reaction chamber 815. Thebody also has a second side comprising valve seats 817. The layer 819can be bonded to the first side of the body, sealing off the opencompartments. The film 821 is capable of transmitting heat and willcover and seal reaction chamber 815. The deformable layer 1303 providesareas that function as diaphragms. When pressed against the second side,for example by a clamping device, the deformable layer 1303 is pressedinto the valve seats, closing the valves and preventing fluid movementthrough fluidic circuits until use.

FIG. 9 shows an embodiment of a cartridge having a fluid distributionchannel. Body 905 comprises a surface on one side that comprises aplurality of fluidic channels 901 oriented in a first direction. Body905 also comprises a surface on a second side comprising a channel 903having an orientation that is oblique to the first direction, forexample, at right angles to the first direction. Channel 903 on a secondside communicates with each of a plurality of fluidic channels on thefirst side through vias 904 that traverse body 905. Channel 903 isclosed by a cover layer 907. Channel 903 optionally communicates with asource of fluid through a bore 920 in piece 905. Fluid channels 901 arecovered by a deformable layer 909. Channels 901 also can comprise valveseats. Portions of the deformable layer can function as diaphragms toopen or close the valves. These can be operated through pneumatic layer911 comprising pneumatic channels that actuate the diaphragms.Alternatively, the cartridge can be engaged with an interface thatprovides an actuation force to the diaphragms.

FIGS. 10A and 10B show a segment of cartridge 1000 having a control lineconfigured to disable a selected diaphragm valve in the cartridge. FIG.10A shows an exploded view of the cartridge. FIG. 10B shows an explodedview in clamshell format. The cartridge comprises a fluidics layer 1001,pneumatic layer 1003 and a deformable layer 1005 sandwiched betweenthem. Fluidics layer 1001 includes at least one fluidic channel 1007that includes recessed valve seat 1009. The fluidics layer alsocomprises a control line 1011 having a branch 1013 that communicatesthrough a via 1015 with a surface of the fluidics layer mated with thedeformable layer. The pneumatic layer comprises a pneumatic channel 1017comprising one or more branches 1019. Each branch controls one diaphragmvalve. The branch includes a valve relief 1021 positioned on thedeformable layer opposite the valve seat in the fluidics layer intowhich the deformable layer can be deformed and which can transmitpressure to the diaphragm, actuating the diaphragm into the valve seat1009. The branch also comprises a valve seat 1023 positioned oppositevia 1015, which via connects to pneumatic control line 1011. Assertionof positive or negative pressure against control line 1011 to thediaphragm actuates the diaphragm against or away from the valve seat1023 in the pneumatic layer. When pressed against the valve seat, thisprevents transmission of pressure through pneumatic actuation channel1017, rendering the diaphragm valve 1009 that controls fluid in fluidicline 1007 inoperative.

FIG. 11 shows a shipping clamp 1103 on cartridge 905.

FIG. 12 shows a cartridge comprising a body 1205 and a reagent reservoir1201. The reagent reservoir has compartments, 1209, that can contain aliquid and that can be sealed with a layer such as heat seal, chemicalseal, adhesive or laser weld. The body can comprise puncturing elements(not shown) to puncture seals in a floor of compartment 1209 when thereagent reservoir is pressed against the body. The puncturing elementscan be a solid material, e.g., that protrude from the body, and that mayhave a tapered end that is pointed or sharp and that is adapted to applyconcentrated pressure to the floor and to puncture a hole in the floor.This creates a fluidic connection between the compartment and a fluidiccircuit in body 1205. This combination also includes flanges 1207 andbreakable tabs 1203. When reagent reservoir 1201 is pressed against body1205, flange 1207 engages tab 1203 and applies a force which breaks tab1203, thereby creating a vent in compartment 1209. This allows fluid incompartment 1209 to drain through the aperture in the floor of thecompartment and into the fluidic circuit.

FIG. 13 shows a diaphragm valve with a thin sealing layer 1303 that cancomprise a heat seal material.

FIG. 14 shows a tool for introducing a dimple into a diaphragm.

FIGS. 15A and 15B show cartridge 1501 having a body 1503 attached to andeformable layer 1505 and having a cover 1507 on the deformable layer sothat the deformable layer in the cartridge is not open-faced. Thiscartridge includes a diaphragm valve 1513 that is normally open and thatregulates fluid flow along a fluidic channel 1515. The cover 1507 coversthe deformable layer and comprises an aperture 1509. The deformablelayer includes a boss 1511 fitted with the aperture. A mechanicalactuator, such as a piston, can be used to close the valve by actuatingthe diaphragm by the provided boss. This cartridge further comprises achamber 1517 for receiving a swab or other sample and a reaction chamber1519 in fluidic communication with channel 1515. The reaction chamber1521 can be covered with a seal and/or can have a separate heat spreaderlayer 1521.

FIGS. 16A and 16B show, respectively, a front and back view of amulti-sample cartridge.

FIG. 17 shows a revolving multi-chamber turret. Cartridge 1701 comprisesa base 1703. The base has a central barrel 1705 comprising a pumpchamber 1707 and movable syringe 1709, a floor 1711 comprising a portstation comprising a port 1713 and a channel 1715 fluidically connectingthe barrel chamber to the port in the floor. The cartridge also has aturret 1717 configured to revolve around the central barrel 1705 andcomprising a plurality of turret chambers (e.g., 1719), each turretchamber comprising a turret chamber aperture 1721, wherein the turret isconfigured to rotate around the syringe barrel, wherein positioning aturret chamber at the port station puts the turret chamber aperture influid communication with the barrel chamber through the floor apertureand wherein the floor covers a turret chamber aperture when turretchamber is positioned at least one position other than port station. Atleast one turret chamber further comprises a channel communicatingbetween the port and an exit port 1723.

FIGS. 18A and 18B shows cross-sectional views of a self-aligning,self-resetting pogo pin 1801 reset into its home position, andfluidically connected to the cartridge, respectively.

FIG. 19 shows an exploded view of an interface slot.

FIG. 20 shows a cartridge interface 1901.

FIG. 21 shows a schematic of a cartridge of this disclosure.

FIG. 22 shows a bottom view of a cartridge circuit.

FIG. 23 shows the valve states and flow for a cell lysis operation.

FIG. 24 shows the valve states and flow for nucleic acid capture.

FIG. 25 shows the valve states and flow for movement of liquid into awaste chamber.

FIG. 26 shows the valve states and flow for creation of a reactionmixture.

FIG. 27 shows the valve states for thermal cycling.

FIG. 28 shows the valve states and flow for movement of theamplification product to output ports.

FIG. 29 shows a cutaway view of a normally open valve actuated by a ramhaving a tip with flexible elements.

FIG. 30 shows a three-dimensional view of a valve of this disclosure.

FIG. 31 shows a valve of this disclosure in a closed configuration. Aflexible end of the ram presses a deformable membrane against a valveseat. The ram is configured to press the deformable membrane so as toseal the valve inlet and the valve outlet by pressure against theperimeter of the inlet and outlet in the valve seat.

FIG. 32 shows a valve having a valve chamber defined by a recess in afluidic layer and a diaphragm comprised in a laminate layer.

DETAILED DESCRIPTION OF THE INVENTION

I. Instruments for Engaging and Operating fluidic Cartridges

In one aspect provided herein is a cartridge comprising: (a) a bodycomprising a fluidic circuit comprising: (1) a sample chamber comprisingan opening configured to receive a sample and a via through the body;(2) a reaction chamber; (3) diaphragm valve elements including a valveinlet and a valve outlet, each configured as a via through the body, anda valve seat; (4) a plurality of ports through the body; and (5) one ormore fluidic channels in a surface of the body, wherein the one or morefluidic channels put the sample chamber (e.g., through the via), thereaction chamber, the valve elements (e.g., through the valve inlet andvalve outlet), and each of the plurality of ports in fluidiccommunication with each other; (b) a cover layer attached to the bodyand sealing the via, the fluidic channels and the reaction chamber; and(c) a deformable layer attached to the body, wherein the deformablelayer (i) comprises a plurality of holes, each hole communicating with aport; and (ii) in combination with the valve inlet, the valve outlet andthe valve seat, form a diaphragm valve configured to regulate fluid flowin the fluidic circuit.

In another aspect provided herein is an instrument comprising: (a) atleast one cartridge interface comprising: (1) an engagement assemblyhaving a first position to receive a cartridge as described herein and asecond position to engage a received cartridge with a manifold assemblyand an optional thermal regulator; (2) a manifold assembly having aplurality of channels, each channel opening onto a front port and a backport, wherein, when the engagement assembly has received a cartridge andis in the second position, a plurality of front ports fluidically engageports in the cartridge and pneumatically engage the diaphragm of thediaphragm valve, and wherein the deformable material serves as a gasketfor fluidic engagement; (3) an optional thermal control assembly (e.g.,thermal cycler) configured to place a heat spreader in thermal contactwith a thermal cycling chamber of an engaged cartridge and to regulatetemperature of the thermal cycling chamber, when the engagement assemblyis the second position; (b) a pneumatic and fluidic assembly comprising:(1) a source of pneumatic pressure; (2) a plurality of fluid sources;(3) a plurality of transmission lines connecting a source of pneumaticpressure in each of the plurality of sources in fluid communication witha back port of the manifold assembly; (4) a pump configured to movefluids from the source through the transmission lines.

FIG. 1 shows an interface configured for a cartridge. Referring to FIGS.4 and 5, for a single sample cartridge, the interface may also includethree fluidic connections 405 and one pneumatic connection 407 tocontrol the valve. These can be low-dead-volume connections whichconnect to the pneumatic and fluidic assembly through tubes.Alternatively, they can be rams such as pogo pins (e.g., 1801 of FIG.18).

This configuration permits samples to be inserted into cartridges, andcartridges to be independently inserted into a slot, even if other slotsare processing other samples. Thus, in one embodiment, the system canprocess samples independently.

The cartridge described in FIGS. 1-5 minimizes the cost of manufactureby minimizing the functions that need to be handled by the disposablecartridge. These functions are moved onto a pneumatic and fluidicassembly, which can be a permanent or semi-permanent part of the system.

In this embodiment, the cartridge can comprise an injection molded body,for example, a plastic, a deformable film; and a foil, such as a metalfoil, each bonded to the body. The body can have integrated alignmentfeatures 209 and 225 so that it can be easily and accurately insertedinto the interface. The plastic material can include any plastic knownto those skilled in the art, such as polypropylene, polystyrene,polyethylene, polyethylene terephthalate, polyester, polyamide,poly(vinylchloride), polycarbonate, polyurethane, polyvinyldienechloride, cyclic olefin copolymer (COC), or any combination thereof.

The cartridge may be scribed with a barcode or QR code for opticalidentification or have an EEPROM or RFID or other similar identificationdevice mounted on the cartridge that can assist in sample tracking andoptionally contain information about the chemistry, process to beperformed, lot number, expiration date, and other information.

The body can have a folding tab 211 that can be snapped shut after theswab is inserted, either by the operator or the system. More than onestyle of body, each adapted to a swab, punch type, or sample type can beproduced. After the tab is snapped shut, the body can serve to containthe sample, providing protection against contamination and facilitatingre-testing or recovery of the sample as required.

The body can also define the volumes for two process chambers. The swab,punch, or other sample type is placed in a compartment 207 that alsoserves as a lysis chamber. To accommodate the swab, punch, or othersample type, it can have a volume ranging from, e.g., 10 μL to 15 ml or1 ml to 10 ml. Cells are lysed and DNA extracted from the swab, punch,or other sample type in this chamber. The second chamber 301, called thereaction chamber, can serve to capture DNA or house a small amount oflysate for direct amplification. It can also be where cleanup and/oramplification occurs. To minimize the duration of thermocycling and theamount of energy required, this second chamber can have minimal volume,perhaps ranging from 2 μl to 25 μl, although other configurations arepractical.

Referring to FIGS. 2 and 3, to an area of the cartridge body 201, adeformable film 203 can be bonded on one side, and a cover sheet 205,such as a plastic film or metal foil, can be bonded to the other.

The deformable material used in cartridges disclosed herein can be aplastic material (plastic deformation) or an elastic material (elasticdeformation). The plastic material can comprise, without limitation, apolymer or a metal. Suitable plastic materials include, withoutlimitation, polypropylene and polyethylene. Suitable metals includealuminum. Suitable elastic materials include, for example, elastomericmaterials such a polysiloxanes, e.g., PDMS. Other deformable materialsare further described herein.

In one embodiment, the deformable film serves as a gasket for threelow-dead-volume connections. These provide an inlet, an outlet, and apurge line that can be used to flush out the cartridge and outlet line.The deformable film also serves as the flexible diaphragm for a valve.The valve seat 305 can be formed into the cartridge body. The inputs 306and outputs 307 to the valve can be vias through the body, channelsbetween the body and the deformable, or both.

The valve can be actuated by positive or negative pressure or applied tothe deformable material over the valve seat. In another embodiment thevalve can be actuated by a ram exerting mechanical force on cover sheet205.

The deformable material may also fill a hole in the body, creating aflexible valve on the far side of the body. The deformable material canbe pressed from the near side to deform through the body, and sealagainst a surface on the far side of the body.

In addition to being mechanically simple, structuring the cartridgearound one molded body with functional elements on the surface increasesthe robustness. Leaks are critical problems, so the fluidic andpneumatic connections need to line up to enable sealing. Tolerancevariations accumulate across assemblies, typically with each partcontributing some variation. As a result, simpler assemblies can be morerobust even with the same part tolerances. Furthermore, the singlesample cartridge and other simplified cartridges in this instantdisclosure involve only a few connections, further reducing the risk ofleakage. Additionally, the effect of thermal expansion increases withsize, so having few connections that are also close together reduces therisks still more.

This embodiment integrates parts, reducing materials and assembly costs.In one embodiment, it is designed with pneumatic ports, fluidic ports,and valve controls (either mechanical or pneumatic) onto one side. Thissimplifies connections and permits more space for other functions suchas a temperature regulator (e.g., a thermocycler) to contact thecartridge, an optical system to interrogate the cartridge, or othermeasurement devices.

Cartridges constructed in this or other embodiments can also be built toaccommodate multiple samples. These multi-sample cartridges can permitthe operator to run multiple samples without having to insert multiplecartridges. (See, e.g., FIGS. 6 and 16)

Alternatively, single-sample cartridges can be assembled ontomulti-cartridge holders. Operators running many samples at a time areable to take the rack containing used cartridges out of the system andinsert the new rack containing unused cartridges. Operators running onlya few samples are able to populate only as many spaces as they wish. Theremaining spaces can be filled with dummy cartridges or left emptydepending on the configuration.

The single-sample cartridges become particularly advantageous whenpaired with a latched cartridge interface 101 that can permit them to beinserted and removed singly. This can provide more flexible sample flow.

This interface can have a number of cartridge positions 103 or ‘slots’that can open and close independently. They can apply a compressiveforce on the cartridges. They can be held closed by solenoids, oranother means controlled by the software, or could be manually latched.

An operator with a new sample to run can insert the sample into acartridge, and snap the cartridge top closed. He or she can then ask thesystem to open a slot. If a slot is not in use, the system opens it,permitting the sample to be inserted. If a processed cartridge is in theslot, the operator removes it. The operator could read the barcode, QRcode, RFID or other identifying material on the cartridge before it isinserted or the system could read as it is inserted or afterwards. Theoperator can then push the interface closed, and enter additionalinformation if necessary. The system can then start processing thesample immediately or start it automatically when next possible.

In an alternative embodiment, an operator with a new sample can manuallyopen a slot or direct the system to open a slot. If a slot is not inuse, the system opens it. If a processed cartridge is in the slot, theoperator removes it. The operator inserts a cartridge. The operatorcould read the barcode, QR code, RFID or other identifying material onthe cartridge before it is inserted or the system could read as it isinserted. The operator could then read the barcode, QR code, RFID orother identifying material on the sample if there is one and place thesample into the cartridge in the slot; alternatively the operator couldenter sample tracking information manually into the system. After thesample has been added, the top of the cartridge can be closed by theoperator or by the instrument. The operator can then push the interfaceclosed manually or the instrument can close the interface. The systemcan then start processing the sample immediately or start itautomatically when next possible.

This instant embodiment can be automated with a loading system thatautomatically inserts and removes cartridges as needed into slots. Theautomation can include mechanisms to load samples from a variety ofsample types such as a collection of tubes containing swabs, microtiterplates containing liquid samples that can include preprocessing fromsingle sources or mixtures, tubes containing liquid samples that caninclude preprocessing from single sources or mixtures, blood containerssuch as Vacutainers, or other containers for additional sample types.

The interface for each cartridge can float, permitting it to seal aroundthe various fluidic and pneumatic connections on one side, whilepressing the cartridge against the thermo regulator (e.g., thermocycler)109 on the other. As shown, the floating interface rotates, pressing thecartridge against a fixed temperature regulator. Alternatively, multiplesmaller thermocyclers could be used. These can rotate or translate,possibly pressing the cartridges against a common pneumatic and fluidicassembly.

After processing, the slot may remain closed to avoid contamination. Theinterface can press the cartridge against a temperature regulator, e.g.,a Peltier device. This contact can be against the foil or film 205enclosing the reaction chamber. Depending on the chemistry used, on theopposite side of the reaction chamber, the interface can house an LED,filter, and photodiode for reaction quantification or another detector.

In such an embodiment, when the reaction is a short tandem repeat (STR)reaction, in many jurisdictions for casework samples, the amount ofhuman DNA must be quantified. The typical forensic process is toquantify an extracted sample using real time polymerase chain reaction(PCR) in a separate instrument before the sample is STR amplified. Inthis instant disclosure, a human specific probe is added to the STRmixture which has fluorescence outside the range used by the STR kit.The reaction chamber 301 is interrogated by a suitable wavelength oflight for the human specific probe while the STR is being PCR amplified.The human specific probe can be a quencher such as a Black HoleQuencher® or a TaqMan® probe or other chemistries well know to oneskilled in the art. As the PCR cycles increase, the fluorescence fromthe human specific probe is monitored to quantify the amount of humanDNA in the reaction. In a preferred embodiment, the number ofamplification cycles can be adjusted based upon the amount of human DNAmeasured; this can be on a cartridge-by-cartridge monitoring ifindependent thermal cyclers are in use. One advantage is that the humanspecific probe will allow the concurrent STR amplification to achieve anoptimal amplification and produce an amount of STR product that isoptimal for the kit regardless of amount of starting DNA in the sample.A second advantage is the real monitoring concurrent with the STRamplification allows integration of a sample-to-answer system withouthaving an additional separate quantification process. A third advantageis for low copy number samples where there is barely enough sample toproduce a good STR profile the integration of the quantification withthe STR amplification prevents the aliquot typically used forquantification from causing the remaining sample to not have enough DNAfor a successful STR amplification.

In addition to actuating the valve diaphragms (e.g., 311, 1301)mechanically, they can be actuated pneumatically. In one embodiment, theinterface 1901 (FIG. 19) provides, for each valve, an interfacediaphragm 1903 that conveys a pressure to the cartridge diaphragm 1301,pushing it against the valve seat 1317 to close the valve. The interfacediaphragm is bonded to the interface block 1905 and encloses a threadedhole 1907 with a fitting 1908 to connect to the flexible tube carryingthe pneumatic signal. Each hole can correspond to a valve, which it canclose or permit to open, controlled by the pneumatic signal. Theinterface diaphragm may be silicone rubber bonded with RTV, with rings1909 to limit delamination from fatigue. However, other deformablematerials can be used.

The interface block 1905 is a component in the interface latchsubassembly 1901. The block has alignment features 1911 and 1913 thatmate to the cartridge alignment features 2003 and 2005 accuratelylocating the cartridge in the interface. The block mounts flexibly to ahinge arm 1915 that pivots to engage the cartridge to the interface, orpermit the operator to insert or remove cartridges. A frame 1917 looselyguides the cartridge during insertion, ensuring that it can mate withthe alignment features.

The single body cartridge allows on-chip storage/integration of reagentreservoir, including, for example, for example, capillaryelectrophoresis separation gel. This embodiment also permits STRmanipulation without having reagents contact PDMS, which can interferewith certain biochemical reactions. This embodiment permits anintegrated reaction chamber: The reaction chamber volume is defined bythe outside of the fluidic layer and enclosed (e.g., by heat sealplastic, heat seal foil, graphite, etc.). It can connect to the circuiteither by vias through the fluidics layer, or by enclosed channels alongthe surface.

In systems that use STR components that are sensitive to PDMS or otherdiaphragm materials, the second side can house the STR components inreservoirs 603 (FIG. 6) and use reaction chambers 605 separate from thePDMS layer 909. To improve room-temperature stability, the STRcomponents can be stored separately. Vias through the fluidics layer maypush or pull the STR components into the reaction chamber, withouthaving the bulk of the STR mixture contact the PDMS or other membranes.

In addition to STR components, other reagents can also be stored on thesecond side of the fluidics layer. For laminated cartridges, which needto maintain a high degree of flatness near the pneumatic and fluidiccircuits, these storage chambers could be above or below the laminate,or off to the side. The storage volumes would need vents near the top,and outlets near the bottom or narrowed sections capable of drawing thefluids upwards like a straw.

To minimize the risk of contamination from one sample to another,reagents that are used before amplification could have separate chambersabove the laminated area. Reagents used after amplification, whencontamination is less of a risk, can be shared among all samplecircuits. This approach permits all reagents needed to run the system tobe stored on a single cartridge.

Those reagents which require low pressures for movement or containmentcan be handled with diaphragm valves. Those reagents which requirehigher pressures, such as the separation gel, can be drawn out at lowpressure into another chamber, and then pushed into the capillaries athigh pressure.

II. Cartridges having a Fluid Distribution Channel

A double-sided fluidic layer offers a number of capabilities formulti-sample cartridges, such as an embodiment as shown in FIG. 6. Forexample, as shown in FIG. 9, if the circuits for individual samples areon one side of the fluidic layer, e.g., through channel 901, the otherside of the fluidic layer could provide right-to-left channels, e.g.,903, to distribute reagents. Reagent distribution can otherwise requirean additional fluidics part or external manifold.

III. Pneumatic Channels to Selectively Block Diaphragm Valves

The right-to-left channels can also route pneumatic control signals toenable or disable specific circuits, as shown in FIG. 10A-B. Thisselective enabling or disabling of circuits can permit some samples tobe run immediately, and other circuits to be reserved to run sampleslater.

IV. Cartridges having a Deformable Layer Sealed to a Plastic Body

Cartridges of this disclosure can have a body comprising a solidmaterial. The solid material can be rigid, plastic (capable ofirreversible deformation) or elastic (capable or reversibledeformation). The body can be stiff or compliant. In some embodiments,the solid material is a polymer, e.g., a thermoplastic, such aspolypropylene. The body can comprise an external surface comprisingelements of fluidic circuits, such as channels, compartments, vias andvalve seats. The body can be made by injection molding of thethermoplastic. These features can be covered with a layer of materialattached to the surface of the cartridge body. The layer can function toseal otherwise open features such as channels and compartments. Thematerial can be a deformable material that can deform to contact a valveseat, thereby closing the valve. In certain embodiments, the solidmaterial is inelastic (not capable of elastic deformation). For example,the solid material is not an elastomer, such as PDMS.

The material can be attached to the surface of the body using aselective bonding process in which the material bonds to selectedportions of the surface during the bonding process and does not bond toun-selected portions of the circuit after the bonding process iscomplete. For example, the material may bond to surfaces other thanfluidic elements during the bonding process, and not bond to fluidicelements, such as channels and valve seats, after the bonding process.Methods for selective bonding include, for example, thermal bonding(e.g., heat sealing, welding, laser welding), chemical bonding (e.g.,chemical bonding of oxide to PDMS) and selectively placed adhesives.

In one embodiment a layer of the deformable material is attached to asurface of a cartridge body through thermal bonding. This can includethermally bonding the material directly to the surface, or thermallybonding the material through an intermediate layer of material. In thelatter case the material can be a laminate in which a deformablematerial is coated with a layer of material that contacts the surfaceand that melts at lower temperature. In either case bonding typicallycomprises contacting the deformable material to the body to form acombination and using a die to apply heat and pressure to thecombination. Application of heat and pressure melts substrates inlocations at which the material and body are in contact and fuse them,e.g., through coalescence. This process is more generally referred to aswelding.

A material that bonds to a body through application of heat and pressureis referred to herein as “heat seal”. Heat seals are well known in theart and are commercially available. For example, 4titude (Walton,Surrey, UK) commercializes a variety of heat seals. These heat seals aredescribed on the website 4ti.co.uk/sealing/heat-seals/. These include,for example, Clear Seal, Clear Weld Seal and Foil Seal. Heat seals alsoare produced by Axygen, a Corning brand (Corning, Tewksbury, Mass.,USA). These include Axygen® PlateMax heat sealing film and sealing filmrolls. See the website: catalog2.corning.com/LifeSciences/en-US/Shopping/Prod uct.aspx?categoryname=Genomics+and+Proteomics(Lifesciences)%7cPCR+Products(Lifesciences)%7cSealing+Films+and+Tapes+for+Microplates(Lifesciences)%7cHeat+Sealing+Films+and+Tapes+for+Microplates(Lifesciences).

The deformable material can be a homogenous or non homogenous material.In certain embodiments, the heat seal material is made from the samematerial as the body of the cartridge. It can comprise a thermoplastic(e.g., polypropylene, polyethylene, polystyrene, cycloolefin co-polymer(COC), mylar, polyacetate) or a metal (e.g., aluminum). See, e.g., WO2012/136333. The heat seal can be produced by contacting a heat seallayer with the body and applying heat and pressure. Non-homogenous filmsinclude laminates having a first side for contact with the heater and asecond side for contact with the body. The first side has higher meltingtemperature (“high melt”) than the second side (“low melt”). Thispermits use of a heat source to bring the lower side to its meltingtemperature before the first side allowing bonding to the body withoutbonding to the heater.

In the single sample cartridge, one side of the body into whichcompartments are formed is covered in a film or foil that can be adheredor thermally attached to the body. This encloses a second functionallayer while only requiring one molded part. This permits functionaldetails—valves, channels, etc. on different sides of the body. In thecase of the single sample cartridge, this permits the valves, pneumaticconnections, and fluidic connections to be on one side of the cartridge,while the reaction chamber is on a different side of the cartridge. As aresult, the temperature regulator controlling the reaction chambertemperature can do so through a thin film, rather than the deformablegasket, which can result in quicker and more controlled thermocycling.

Referring to FIG. 13, in this embodiment, the valve diaphragms 1301 areformed by a film, such as a plastic film. These films are sealed to thecartridge body 1311, enclosing the fluidic circuit 1315. The sealing canbe through a heat-seal, a pressure-seal, laser welding, chemicalbonding, adhesive or other method well known to one skilled in the art.These valves can be actuated by a control circuit on the system 1305.However, the control circuit can be a permanent part of the systeminterface 1309 and need not be part of the disposable cartridge 1311.This control circuit can be mounted to a mechanical support plate, withthrough vias to conduct the pneumatic signals. Gaskets 1313 between thesupport plate 1307, control circuit 1305, and the cartridge will preventleaks. In one embodiment the gaskets 1313 can be part of the interface1309. In an alternative embodiment the gaskets 1313 can be part of thedisposable cartridge 1311.

Depending on the film used, there can be a slight overhang around theperimeter of the valve, channel, or volume. This overhang can be due toadhesive or plastic flow during bonding. To prevent these from affectingthe quality of the valve seal, the valve inlet, outlet, or both can bethrough vias in the valve seat 1317. The valve seat, away from theperimeter, can be less affected by the overhang.

Because of the limited flexibility of the film, it may be necessary tocreate a dimple over the valves. This can be achieved by coining thefilm downwards against the valve, with the limitation that the heatapplied must not be enough to bond the film to the valve seat. Apreferred approach would be to vacuform the dimples. The ordinaryprocess of heat sealing involves applying a combination of heat andpressure to create a bond. If the heated tool (1401) was made from aporous material and had cavities cut above the valves, suction could beapplied that can draw the film over the valve into the cavity, creatinga dimple. This can occur at the same time as the film was being bondedto the body in other areas.

This embodiment can allow multiple fabrication and material options. Forexample, PDMS, which is commonly used in microfluidics, could bereplaced with such a material, such as the heat seal films. Thisembodiment also reduces requirement for flatness in pieces, permittingother cartridge materials, such as polypropylene.

The use of the fluidics layer for reagent storage and the use ofsections of the enclosing film for shipping as in the embodiment of 601,and the use of sections of the enclosing film to implement valves as inthe embodiment of 701, permits the cartridge functions to be served byone molded piece and one or more bonded films. Another embodiment, asshown in FIG. 8, uses this construction.

V. Clamp-Sealed Cartridges

FIG. 11 shows a section of the cartridge 601. By using raised lines orareas, e.g., ridges, 1101 to control the bonding of the film, vents canbe built into the fluidic layer 905. These raised areas can provide alocalized contact when bonding, controlling which areas are bonded andwhich areas are not, resulting in defined channels.

To close off the outlets 1109 and vents 1105 to these reagent chambers1107, bar clamps 1103 can be built into the shipping container for thecartridge. These bar clamps can have some rigidity, but can be coveredby a deformable or other material that can conform to the cartridgesurface. It may have a basic shape or be formed to mate with thecartridge surface.

Bar clamp 1103 is able to hold the seal film cover (907, not shown inFIG. 11) against the body or fluidic layer 905, closing off the definedchannels. After shipment but before use, the cartridge is removed fromits packaging, which either removes the shipping clamps as the packagingis opened, or the clamps are removed separately from the cartridge afterthe cartridge is removed from the packaging.

If the flexible bar clamp is U-shaped as shown, it can close eachchannel in two places to prevent leakage. The operator will then be ableto confirm that no leakage has taken place by examining the area betweenthe two seals. Any leakage past both seals will generally leave aresidual amount between the two seals.

Before use, the two vias leading to each of the reagent reservoirs areheld closed by a shipping clamp. This shipping clamp can apply a uniformforce to a flexible pad, causing the pad to deform and hold the valvesclosed. Alternatively, it can include a number of small rubber contactsthat can individually hold each valve closed. This shipping clamp canthen be removed before the cartridge is inserted into the system.

VI. Diaphragm Valve with a Bossed Diaphragm

Diaphragm valves also can be actuated mechanically using a ram, e.g., apin. These can be actuated by a solenoid. If actuated by solenoid, itmay be beneficial to add a boss (such as element 1511) to the outside ofthe deformable. This permits a ram to push against the boss, creating acentered force sealing the valve, even if the solenoid is not centeredover the valve.

VII. Turret Cartridge

Cartridges actuated mainly by a syringe pump or by a manually operatedsyringe are included in this instant disclosure. The cartridges can becontrolled by motors controlled by the computer on the system.

One embodiment of a cartridge utilizes a syringe pump for actuation,with selectable, specialized areas arranged in a ring. These areas caneach store reagents, house the swab or punch, contact a temperatureregulator, connect to the capillary for separation, etc.

Referring to FIG. 17, the cartridge can implement a rotary selectorvalve, either by rotating the cartridge body 1717 or an internal valve.By rotating, various inputs or outputs can be selected. This rotationcan be driven by, for example, a stepper motor. The syringe 1709 can inturn be driven by for example, a linear stepper motor. This permits abroad range of general functions to be controlled by two stepper motors.The interface can also make use of one or more temperature regulators.Thermocycling can be implemented by cycling the temperature of atemperature regulator, or by rotating to contact one of multiplecontrolled heat sources to reduce the power usage and may increasethermocycling speed. It can also have an LED, filter, and photodiode forreaction quantification.

One, two or three positions on the hub can be temperature controlled.One position on the hub can be open on top, for sample insertion. One ormore positions can have external, retractable magnets.

Turret cambers can include: (A) Vent: air to injection chamber; (B)Vent: to denature heater; (C) lysis chamber/swab vial; (D) lysisbuffer/Waste; (E) mix chamber/beads; (F) water; (G) ethanol; (H) STRlyosphere (amplification reagents); (I) capture solution and sizestandard (or lyosphere); (J) eluting agent; (K) electrophoresisseparation gel; (L) reaction chamber.

Gel injection may be to a booster pump instead of directly to thecapillary. This would avoid the need for high-pressure seals, in thesample cartridge (This would permit gel injection in parallel with otherfunctions.) If the capillary can be mounted directly, denature headingmay be complete by one of the heated positions, without an externaldenature heater. An external waste gate, at the cathode end of thecapillary may still be necessary.

This embodiment can permit an interface consisting of one rotary and onestepper motor, eliminating pneumatic pumps, manifolds, anode module/gelfilling mechanism, etc.

VIII. Lead-in Guiding Fluid Delivery Pogo Pin

FIGS. 18A and 18B show a low-dead-volume floating connector 1808 incross-section. When the interface is open as shown in FIG. 18A, the pogois forced down against the home lead-in 1801 by a spring 1802. This willreset the pin to a consistent home position relative to the pogo block1803. When the interface closes onto a cartridge that is off-center, thepogo contacts the engagement lead-in 1804 and is pushed up, freeing theengagement play 1805. The engagement lead-in then guides the cartridgewithin this play. Once aligned, the conic surfaces of the pogo pin 1806and cartridge 1807 connect. The slight taper magnifies the force of thespring, creating a seal. This seal requires some flexibility in thecartridge. Since the surrounding wall thickness is driven by theengagement lead-in, this limits the lead-in size. The engagement lead-in1804 and the engagement play 1805 will both need to be large enough toaccommodate all manufacturing and other tolerance variations. If thepogo pins did not self-reset to a consistent home position, theengagement lead-in can changed to accommodate variations from theengagement play as well.

IX. Vent Tabs

In an embodiment shown in FIG. 12, the cartridge includes two injectionmolded plastic parts, a cartridge body and a reagent reservoir. When inuse, the reagent reservoir can be pressed against the body. This cansnap open the vents, and engage connections between the body and thereservoir.

For cartridge concepts that have bodies 1205 and moveable reagentreservoirs 1201, such as the cartridge of the embodiment of 701, thereis another approach to providing vents. This approach does not requireadditional parts. This is to build in designed-to-fail tabs (e.g., 1203)into the reagent reservoir. Before use, these tabs will remain closed,but will have a slight interference with the cartridge body 1205. Whenthe reagent reservoir is engaged by pressing, these tabs will try topull away from the main volume. It will tear or crack, opening a smallvent in the reservoir.

This embodiment can provide a vent for on-cartridge reagent reservoirswithout requiring additional degree of freedom in the interface oradditional part in the cartridge.

X. Fluidic Device with Diaphragm Valve

The cartridge can utilize off-cartridge pumps to move liquids. To avoidthe need for high mechanical precision, these valves and channels can belarger than traditional microfluidic valves and channels.

The cartridge of this disclosure can include diaphragm valves. Diaphragmvalve can be formed having a valve chamber in the fluidics layer of thecartridge and a deformable membrane attached to a surface of thefluidics layer and providing a diaphragm for opening and closing valve.In one embodiment, the valves are normally open. That is, at ambientpressure the valve is open and closing the valve involves applyingpositive pressure to the diaphragm opposite the valve seat. Applyingnegative pressure to the diaphragm opposite the valve seat can furtheropen the valve. The diaphragm can be actuated by pneumatic or mechanicalpressure. In an embodiment of this disclosure the diaphragm ismechanically actuated by positive pressure applied by a ram or rodhaving an end configured for insertion into the valve chamber. Incertain embodiments the rod has a compliant end that promotes contactbetween the diaphragm and a valve seat, thereby sealing the valveclosed. Withdrawal of the rod relieves pressure on the diaphragm,thereby opening the valve.

In one embodiment of a normally open valve, a surface of the fluidicslayer comprises a recess that both defines a valve chamber and functionsas a valve seat. At ambient pressure the deformable membrane does notsit against the valve seat and the valve is in an open configuration.Positive pressure on the deformable membrane from the side opposite thefluidics layer pushes the deformable membrane against the valve seat,closing the valve. The valve seat can take a curved shape that is convexwith respect to the surface of the fluidic layer, against which thedeformable membrane can conform. For example, the valve shape can be asection of a sphere or an inverted dimple or a dome. Such aconfiguration decreases the dead volume of the valve, e.g., by notincluding a valve chamber that contains liquid while the valve isclosed. This valve also comprises a surface against which the deformablemembrane can conform easily to close the valve. In another embodiment,the concave surface can comprise within it a sub-section having a convexsurface, e.g., an inverted dimple comprising an extraverted dimplewithin it forming, for example, a saddle shape. The convex area rises upto meet the deformable membrane under pressure, creating a better sealfor the valve.

Valve seats can be recessed with respect to the rest of the surface byabout 25 microns to about 1000 microns, e.g., about 700 microns. Valvescan communicate with fluidic channels that are either microfluidic ormacrofluidic (e.g., having an aspect less than 500 microns or having anaspect greater than 500 microns or at least 1000 microns). In certainembodiments of a normally open valve, the concavity is recessed lessthan the channels to which it is connected. In certain embodiments thechannels can enter partially into the concavity, for example forming avault. In certain embodiments, the channels and concavity are formed bymicromachining, injection molding or embossing.

XI. Valve Actuated by Ram with Compliant End

One embodiment involves closing a fluidic valve with a ram. The valvecan be comprised in substrate that contains the valve and one or moreinput and output fluidic channels. There can be more than one input andoutput. These channels can enter the surface of the dome valve at anylocation on the surface as long as there is a sealing surface betweenchannels. In certain embodiments, channels can enter the valve chamberthrough vias that connect with the channels. The dome valve is thencovered with a membrane either elastic or non-elastic film. The film isaffixed to the perimeter of the dome to create an air and liquid tightseal. The ram is then pressed against the film diaphragm with sufficientforce to deform the diaphragm and press the film onto the dome surface.The pressure from the ram creates a fluidic seal between the orifices ofthe ports entering the dome valve.

In one embodiment the valve is configured as a router. The router canhave, for example, four inlets/outlets. In this configuration the forkedram, when engaged, can block access to the router by some, but not all,of the inlets/outlets. For example, the forked ram could allow fluidflow through the router in a north-south direction or not it in anEast-West direction.

The ram is structured such that there are one or more flexure postsdefining an identical dome surface to match the valve dome surface withthe offset of the thickness of the diaphragm. The flexure posts with theseal seat tips will be able to self align to the target seal areas ofthe dome, namely the perimeter of the orifice for the input and outputchannels of the valve. The flexure posts also concentrate the stressgenerated by the force applied to the overall post onto the active sealarea.

Referring to FIGS. 29 and 30, a fluid chip body comprises a recessforming a valve seat (“dome valve”). The recess defines a space thatfunctions as a valve chamber. The fluidic chip body also includesfluidic channels (which can be microfluidic channels) in fluidcommunication with the valve through inlets and outlets. A surface of afluidic chip body into which the recesses impose is overlaid with adeformable membrane (“elastic film”). A ram actuates the valve byapplying pressure to deformable membrane. The ram can include a fork orslotted end that provides compliance to the flexure posts tines of thefork. An end of the ram has a form that complies with the shape of thevalve seat. Referring to FIG. 31, when pressed against the deformablemembrane, the ram deforms the deformable membrane against the valveseat. By contacting the valve seat around the valve inlet and valveoutlet, the diaphragm closes the valve, preventing fluid flow throughthe valve. Relieving pressure on the diaphragm by withdrawing the ramallows the deformable membrane to assume its neutral position, openingthe valve to fluid flow. The ram can be actuated, for example, by asolenoid.

XII. Reaction Chamber

In one embodiment a fluidic device of this disclosure comprises areaction chamber that comprises a solid substrate, e.g., solid phaseextraction material, for retaining analyte from the sample. The solidsubstrate can comprise a material that binds the analyte, such as anucleic acid such as DNA. The amount of solid substrate in a chamber andthe selected to retain the predefined amount of analyte. For example,the material can be a Whatman FTA paper or a carboxylated material.Alternatively, the solid substrate can be an absorbent or sponge-likematerial that absorbs a predetermined volume of fluid. The material canbe in the form of a monolith. The material can be, for example, PVDF(polyvinyldiene fluoride) membranes, filter paper, glass particles,silica, or other solid phase extraction material. In operation, lysateis pumped through the chamber and a predetermined amount of analyte isretained on a solid substrate. Then, retained material is contacted withreagents, e.g., reagents for PCR. The resulting material can beincubated to form a reaction product. For example, the chamber can beput into thermal contact with a thermal-control device, such as aPeltier, and the reaction mixture can be thermal cycled. In anotherembodiment, the chamber can include a pocket or container designed toretain the defined volume of liquid.

XIII. Contaminant Deactivation

In one embodiment the fluidic layer includes a waste chamber. A wastechamber can contain material that degrades nucleic acids, polypeptides,or other analytes. For example a material can comprise a chlorinatedmaterial, such as calcium hypochlorite. Alternatively, the waste chambercan include in absorbent material that absorbs waste containing liquid

In another embodiment the nucleic acid degrading material is containedin a water-soluble capsules in yet another embodiment the nucleic aciddegrading material is combined with an absorbent material such ascellulose or polypropylene fibers.

In another embodiment, the waste chamber contains enzymes that degradethe nucleic acids (e.g., nucleases), polypeptides (e.g., proteases), orother analytes such as phosphorylated sites (e.g., phosphatases).

XIV. Cartridge and Method

FIGS. 21 and 22 shows a fluidic cartridge configured for extractingnucleic acid from a sample, performing amplification on the sample, andoutputting the amplification product. The cartridge includes a portconfigured to accept a sample container adapted to receive a sample,such as a swab; a port configured to accept a syringe pump containing orconnected to reagents, such a lysis solution; a port configured toaccept receptacles separately carrying PCR master mix and PCR primers; areaction chamber, e.g., for thermal cycling; a waste chamber; a vent; anoutput port; fluidic channels (which can be microfluidic or microfluidicchannels) in communication with these elements; valves for regulatingflow of fluids in the fluidics circuit all of the cartridge. The valvescan be, for example, diaphragm valves.

FIG. 23 shows the operation of a cartridge. Closed valves are indicatedin darker shade, open valves are indicated in lighter shade. Arrowsindicate the flow of liquids which are moved by the syringe. Lysissolution from the syringe is moved through a fluidic channel into thecontainer containing a sample. The sample can be heated or sonicated tofacilitate cell lysis. In FIG. 24 lysate is pulled back into the syringeand nucleic acid is captured on a solid phase in the reaction chamber.In FIG. 25 lysis solution is transported into the waste chamber. In FIG.26 PCR master mix and primers, which can be contained in separatecontainers, are moved into the reaction chamber, for example pushing theliquid from one side as the syringe provides vacuum from another side.In FIG. 27, the reaction chamber is subjected to thermal cycling toamplify target sequences, for example, STR sequences, while all thevalves are closed. In FIG. 28 the amplification product is moved to anoutput port where it can be transferred for further analysis.

XV. Integrated System

The cartridges of this disclosure are useful in integrated and automatedsample-to-answer systems that, starting from a sample comprisingbiological material, generate an analysis of the sample. In certainembodiments, the biological material is DNA and the genetic profileinvolves determining one or a plurality of alleles at one or a pluralityof loci (e.g., genetic loci) of a subject, for example, a STR (shorttandem repeat) profile, for example as used in the CODIS system. Thesystem can perform several operations, including (a) extraction andisolation of nucleic acid; (b) amplification of nucleotide sequences atselected loci (e.g., genetic loci); and (c) detection and analysis ofamplification product. These operations can be carried out in a systemthat comprises several integrated modules, including an analytepreparation module; a detection and analysis module and a controlmodule.

Systems provided herein may be fully integrated. Sample processing canbe accomplished in a single system without having to remove a sample andtransfer it to another system. Systems provided herein can be fullyautomated, enabling a user to process a sample without substantial inputfrom the user.

A sample preparation module includes a cartridge module assemblyconfigured to engage and operate one or more than one sample cartridge.A sample cartridge is configured to receive one or more samples and toperform nucleic acid extraction and isolation, and DNA amplificationwhen the cartridge is engaged with a cartridge module assembly in thesystem. It can also include controls and standards for assisting inanalysis. In other embodiments, a sample cartridge is configured toreceive one or more samples and to perform cell lysis, and enzymaticassays when the cartridge is engaged with a cartridge module assembly inthe system.

The sample preparation module can include a receptacle for receiving oneor more cartridges, an engagement assembly to engage the cartridge; afluidic manifold configured to engage ports in a cartridge and todeliver pressure and/or fluids to the cartridge through the ports; adelivery assembly configured to deliver reagents, such as amplificationpre-mix, from a compartment in the sample cartridge to an amplificationcompartment; a pneumatic manifold configured to engage ports in acartridge and to deliver positive or negative pressure to the cartridgethrough the ports for moving fluids and operating valves, pumps androuters in the cartridge; a pump configured to deliver pressure to thefluidic and pneumatic manifold. Consumable reagents can be carried in amodule, e.g., a buffer module, that is, removably engageable with thecartridge module.

PCR can be carried out using a thermal cycler assembly. This assemblycan include thermal controller, such as a Peltier device, infraredradiation source, resistive heating element, circulating water or otherfluids, circulating air, movement of constant temperature blocks, orother material, which can be configured to heat and cool for thermalcycling and can be comprised in the cartridge module which can beconfigured to move the thermal controller into thermal contact with thethermal cycling chambers, for example, through a heat spreader (orthermoconductor that can spread/distribute heat and cooling) disposedover each of the reaction chambers. In some embodiments, the cartridgecomprises a temperature regulator assembly having one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 16, 24, 32, 40, 48 or more) thermocyclingchambers and the sample cartridge can be in fluid communication with afluidic channel.

An analysis and detection module is configured to receive analyte fromthe sample preparation module and perform capillary electrophoresis onthe analyte to detect analytes separated by electrophoresis and toanalyze the detected analytes. It can include a capillaryelectrophoresis assembly, a detection assembly, and an analysisassembly.

The capillary electrophoresis assembly can include an injectionassembly, that can include a denature heater assembly, a positioningassembly for positioning an analyte for capillary injection; a cathodeassembly; a capillary assembly; an anode assembly; a capillary fillingassembly for filling a capillary with separation medium and a powersource for applying a voltage between the anode and the cathode.

A detection assembly can comprise a laser configured to illuminate thecapillaries and a detector. The laser can be configured to excitefluorescent dyes in the analyte. In alternative embodiments, the lasercan be replaced by an alternate light source such as an LED. Thedetector can include a CCD array, photomultiplier, diode array, or otherdetector, for detecting light produced by excited dyes and for producingan output signal.

An analysis assembly can include a computer comprising memory and aprocessor for executing code (e.g., code on a tangible medium) foranalyzing the output signal and producing a computer file containing ananalysis of the signal. Such an analysis can include, for example,identification of alleles from various STR loci. The computer file canbe in a format that is compatible with public databases. For example,the file can be in CODIS format which is compatible with the NationalDNA Index System (NDIS) operated by the FBI.

The system can be operated by a control module. The control module caninclude a user interface configured to receive instructions from anddeliver information to a user. It can include software programmed toexecute routines for performing the operations mentioned, above, andtransmit and receive information, such as computer files, from remotelocations, for example, over the internet.

XVI. Method of Use

The cartridges of this disclosure can be used in an integrated systemfor preparing a sample, for example, DNA isolation and amplification.For example, in one embodiment, a sample contained on for example a swabor a card punch, can be introduced into sample chamber 207. The chambercan be snapped shut by the lid 211. The cartridge can be engaged withcartridge interface 103. Cell lysis buffer contained in an on-systemreservoir can be feed through line 405 through interface assembly 401into the fluidic channel in the cartridge and into the sample chamber207. After lysis, lysate can be moved through a fluidic channel on thechip, for example, which pumps the fluid into a reaction chamber 301. Inone embodiment, the DNA reaction chamber can include magneticallyattractable particles that bind DNA and that can be immobilized in thereaction chamber by applying a magnetic force generated in theinterface. This can eliminate the need for an intermediate DNA isolationchamber. Waste fluid can be moved through the cartridge and out througha vent. Reagents for performing PCR or other reactions can introducedinto the reaction chamber through one of the fluid lines 405 connectedto the interface. A thermal control mechanism in the system can applyheat to perform thermal cycling in a thermal cycling chamber 301 of thecartridge. In some embodiments the heat is applied to a heattransmission element, for example, a foil or metalized film, thatimproves thermal contact and transmission.

The cartridges of this disclosure can be used in an integrated systemfor analyzing a sample, for example, DNA isolation and amplificationwith real time or end point detection. For real time measurement, thesamples can be interrogated by an optical detection system whileamplifying in reaction chamber 301. The readout can be the change influorescence or by melting point. The probes can be human specific forhuman identification, forensics, or molecular diagnostic applications,or specific for pathogens for molecular diagnostic applications, or forbioagents for biodefense applications or nonspecific intercalators fordetermining amount of DNA present. Amplification methods include, forexample, thermal or isothermal amplification reactions, for example,PCR, rolling circle amplification, whole genome amplification, nucleicacid sequence-based amplification, and single strand displacementamplification, single primer isothermal linear amplification (SPIA),loop-mediated isothermal amplification, ligation-mediated rolling circleamplification and the like

The cartridges of this disclosure can be used in an integrated systemfor analyzing a sample. The assay can detect a polypeptide (e.g.,immunoassay) or a nucleic acid (e.g., PCR). The assay can be multiplexor single analyte. They can involve any assay to measure presence,amount, activity, or other characteristics of the sample. These includeassays that involve detection by fluorescence, luminescence,chemiluminescence, Raman, absorbance, reflectance, transmittance,birefringence, refractive index, colorimetric and combinations thereof.In this instant disclosure, the enzyme master mix and the substratemight be individually added to the reaction and the progress or endpointof the assay monitored optically.

For STR applications, after thermal cycling, other reagents such asmolecular weight markers (size standards) can be combined with the PCRproduct. Movement through the cartridge can be controlled when diaphragmvalve 303 is actuated by pneumatic or mechanical actuators whereinforces transmitted through line 407. Products of the PCR can be movedoff chip for analysis through an output line.

While preferred claims of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch claims are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the claims of the invention described herein maybe employed in practicing the invention. It is intended that thefollowing claims define the scope of the invention and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

1. A system, comprising a cartridge and a cartridge interface configuredto receive the cartridge, the cartridge comprising: a cartridge bodyhaving a valve seat recessed into a surface of the cartridge body; and adeformable layer thermally bonded to the surface of the cartridge bodyand covering the valve seat, wherein the deformable layer comprises anon-homogenous material having a first side configured to contact aheater and a second side contacting the body, the first side having ahigher melting temperature than the second side; and the cartridgeinterface being further configured to supply positive pressure ornegative pressure to at least a portion of the deformable layer coveringthe valve seat.
 2. A method of operating the system of claim 1, themethod comprising: engaging the cartridge with the cartridge interface;and supplying positive pressure or negative pressure to at least aportion of the deformable layer covering the valve seat.
 3. A method ofactuating a valve in a cartridge system, the method comprising: applyingpositive pressure or negative pressure to at least one portion of adeformable layer that is thermally bonded to a surface of a cartridgebody and covers a valve seat recessed into the surface of the cartridgebody, thereby actuating the deformable material into or out of contactwith the valve seat, wherein the deformable layer comprises anon-homogenous material having a first side configured to contact aheater and a second side contacting the body, the first side having ahigher melting temperature than the second side.
 4. The method of claim3 further comprising engaging the cartridge body with a cartridgeinterface configured to supply the positive pressure or the negativepressure to the at least one portion of the deformable layer.
 5. Acartridge comprising: a cartridge body having a valve seat recessed intoa surface of the cartridge body; and a deformable layer thermally bondedto the surface of the cartridge body and covering the valve seat,wherein the deformable layer comprises a non-homogenous material havinga first side configured to contact a heater and a second side contactingthe body, the first side having a higher melting temperature than thesecond side.
 6. The cartridge of claim 5, wherein the non-homogenousmaterial comprises a laminate.
 7. The cartridge of claim 5, wherein thenon-homogenous material is not an elastomeric material.
 8. The cartridgeof claim 5, wherein non-homogenous material is structurally configuredfor plastic deformation.
 9. The cartridge of claim 5, wherein at leastone portion of the deformable layer comprises a permanent deformation.10. The cartridge of claim 5, wherein the valve seat has a curved shapethat is concave with respect to the surface.
 11. The cartridge of claim5 further comprising a valve inlet and a valve outlet on opposing sidesof the valve seat and a fluid channel in fluidic communication with thevalve inlet and the valve outlet.
 12. The cartridge of claim 11 furthercomprising a reaction chamber in fluidic communication with the fluidchannel.
 13. A method, comprising engaging the cartridge of claim 5 witha cartridge interface configured to supply positive pressure or negativepressure to at least a portion of the deformable layer covering thevalve seat.
 14. The method of claim 13 further comprising supplyingpositive pressure or negative pressure to at least a portion of thedeformable layer covering the valve seat, thereby actuating thedeformable layer into or out of contact with the valve seat.
 15. Amethod of using the cartridge of claim 5, the method comprising: (a)providing a reaction mixture comprising: (i) a sample comprisingmammalian DNA; (ii) reagents for amplifying short tandem repeats (STRs)in the mammalian DNA; and (iii) a mammalian-specific probe selected tobe amplified in a STR reaction, the mammalian-specific probe including alabel that is distinguishable from the labeled primers; (b) performingthe STR reaction in the cartridge of claim 5, wherein performing the STRreaction comprises: (i) amplifying the STRs in the mammalian DNA; and(ii) amplifying the mammalian-specific probe; (c) detecting an amount ofamplified mammalian-specific probe in the reaction; and (d) optionally,stopping the STR reaction based on an amount of amplifiedmammalian-specific probe detected.
 16. The method of claim 15, whereinthe reagents comprise one or more of labeled primers, nucleotides, andpolymerase.
 17. The method of claim 15, wherein detecting an amount ofamplified mammalian specific probe in the reaction occurs over a firstperiod of time.
 18. The method of claim 15, wherein detecting an amountof amplified mammalian specific probe in the reaction comprisesperforming real-time PCR.
 19. The method of claim 15 wherein the labelsare fluorescent labels and the distinguishable label has a wavelengthabove or below the highest or lowest wavelength of labeled primers. 20.A method comprising: (a) providing a reaction mixture comprising: (i) asample comprising mammalian DNA; (ii) reagents for amplifying shorttandem repeats (STRs) in the mammalian DNA; and (iii) amammalian-specific probe selected to be amplified in a STR reaction, themammalian-specific probe including a label that is distinguishable fromthe labeled primers; (b) performing the STR reaction comprising: (i)amplifying the STRs in the mammalian DNA; and (ii) amplifying themammalian-specific probe; (c) detecting an amount of amplifiedmammalian-specific probe in the STR reaction; and (d) optionally,stopping the STR reaction based on an amount of amplifiedmammalian-specific probe detected.